Project Summary/Abstract Despite recent gains, the burden of malaria caused by P. falciparum (Pf) in sub-Saharan Africa remains unacceptably high. Vaccines offer the greatest potential to produce sustained protection against malaria, but the only licensed vaccine for malaria is short lived and only modestly effective. Decades of research have been unable to produce a feasible vaccine which approaches the efficacy of naturally acquired immunity (NAI), which provides nearly complete protection against symptomatic disease in adults in many endemic areas. NAI to malaria is complex slow to develop. Antibodies are key effectors of NAI, and eventually overcome the high degree of genetic diversity evolved by Pf antigens important in host-parasite interactions. This diversity may underlie the slow development of NAI and relative inefficacy of current vaccines. While it is loosely understood that increasing cumulative exposure to Pf leads to greater immunity, fundamental questions remain unanswered. The aims of this project are (1) to determine the effect of the number, timing, and genetic composition of blood stage Pf infections on NAI in childhood; and (2) to determine the effect of the number, timing, and genetic composition of exposure to Pf in childhood on the antibody repertoire, and how antibody responses relate to immunity. We are in a unique position to achieve these aims by leveraging an extensive set of existing samples and data from multiple intensively followed early childhood cohorts in Uganda along with a team of experienced collaborators with expertise in malaria epidemiology, statistics, genomics, and immunology. In Aim 1, we will initially perform whole genome sequencing on a subset of Pf infections in our cohorts to systematically identify antigenic variants under potential selection. These antigens, along with others identified from existing data, will be deep sequenced from all infected time points using highly multiplexed deep sequencing. We will quantify anti-parasite and anti-disease immunity using detailed clinical data, and combine these data to test 3 fundamental hypotheses regarding the relationship between exposure and immunity. In Aim 2, we will longitudinally evaluate antibody responses to Pf in our cohorts using ultra-high throughput long peptide arrays, targeting the entire genome including all identified antigenic variants. We will identify sequences of key antigens from these experiments and from Aim 1, and further evaluate antibody responses using custom protein arrays with experimental validation using functional assays. Using these data, we will test an additional 3 hypotheses. At the conclusion of this project, we will have identified fundamental factors driving the development of NAI, including the role of strain-specific vs. transcendent immunity and the identification of antigenic variants most associated with immunity and thus most appropriate for inclusion in a malaria vaccine.